EP3164500B1 - Low gluten yeast hydrolysates - Google Patents

Low gluten yeast hydrolysates Download PDF

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EP3164500B1
EP3164500B1 EP15732638.0A EP15732638A EP3164500B1 EP 3164500 B1 EP3164500 B1 EP 3164500B1 EP 15732638 A EP15732638 A EP 15732638A EP 3164500 B1 EP3164500 B1 EP 3164500B1
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yeast
hydrolysate
ppm
gluten
dry matter
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French (fr)
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EP3164500A1 (en
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VAN Petrus Norbertus BEELEN
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DSM IP Assets BV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/347—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of proteins from microorganisms or unicellular algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A23L33/145—Extracts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21026—Prolyl oligopeptidase (3.4.21.26), i.e. proline-specific endopeptidase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to low gluten yeast hydrolysates and a process for the for producing the same using a prolyl-specific endoprotease.
  • yeast extract typically baker's and torula yeast cells may be subjected to autolysis.
  • Some sources of yeast e.g. spent brewer's yeast
  • yeast residues of gluten
  • extracts and suspensions derived of yeasts sources containing gliadins have limited use in food applications due to the intolerance towards gliadins in part of the population (Celiac disease).
  • typical yeast extracts or autolysates obtained from sources containing gliadins e.g. brewer's yeast extracts
  • typical yeast extracts or autolysates obtained from sources containing gliadins contain gliadins in a concentration exceeding the acceptable threshold of 20 ppm by weight.
  • gliadins Reduction of gliadins from yeast sources containing gliadins enables the use of these yeasts and extracts thereof is food applications which require absence of low concentrations of gluten.
  • a problem with the reduction of gliadins in yeast is to reduce specifically the gliadin content without undesired side effects of the treatment. For example, it is undesired that the treatment aiming to reduce the gliadin content also degrades the yeast' RNA.
  • US5286630 discloses yeast extracts from Candida utilis which can be used as flavor enhancers and emulsifiers.
  • the extract is isolated by heating Candida utilis cells, rupturing the cells, separating the ruptured and recovering the soluble yeast extract.
  • WO2005/027953 discloses the use of proline specific endoproteases to hydrolyse peptides and proteins comprising 4 to 40 amino acid residues.
  • WO2005/067734 discloses a process to produce a composition containing 5'-ribonucleotides wherein a microorganism is subjected to autolysis under conditions at which a substantial part of the RNA remains in a form degradable into 5'-ribonucleotides and at which a substantial part of the RNA remains associated with the cell wall fraction.
  • WO2012/110534 discloses a process to produce a yeast extract comprising a) subjecting yeast to autolysis, and b) subjecting the autolysate to solid/liquid separation and recovering the soluble fraction, whereby the solid liquid separation in step b) is done at a pH of less than 5.1.
  • JP2007 049988 discloses the provision of yeast extracts by the use of a protease, a 5'-phosphodiesterase and 5'-adelyatedeaminase, resulting in an increase in the peptide and nucleic acid content of the hydrolysates.
  • EP2256184 describes a Saccharomyces cerevisiae mutant strain having a RNA content of 14% by weight or more based on the dry cell weight and a method for producing a yeast of high RNA content by using the strain.
  • gliadins can be removed from a yeast source containing gliadins by incubation with a proline-specific endoprotease during the hydrolysis and/or autolysis process. It was surprisingly found that the gliadins can effectively be hydrolyzed to a level below 20 ppm by weight on dry matter content of the yeast autolysates and extracts. Further, it was found that incubation with a proline-specific endoprotease during the hydrolysis and/or autolysis process did not degrade the RNA present in the yeast.
  • yeast is defined herein as a solid, paste or liquid composition comprising yeast cells.
  • the yeast cells are from the genus Saccharomyces.
  • the yeast may be produced in a fermentation process such as a process for the production common baker's yeast.
  • the yeast is a brewer's yeast, such as a spent brewer's yeast that may be obtained as a side stream from beer brewing processes.
  • Autolysis is defined herein as the enzymatic breakdown of yeast cells using endogenous yeast enzymes and optionally exogenous added enzymes. Autolysis may result in both a yeast autolysate and a yeast extract (see definitions below).
  • Hydrolysis is defined herein as the enzymatic breakdown of yeast cells using only exogenous enzymes. The endogenous yeast enzymes are first inactivated for instance by a heat shock. Hydrolysis may also result in both a yeast autolysate and a yeast extract (see definitions below).
  • yeast hydrolysate is defined herein as the digest of yeast, such as a spent brewer's yeast, obtained by autolysis or hydrolysis as defined herein before and resulting in a yeast autolysate or a yeast extract as defined herein below.
  • yeast autolysate is the concentrated, not extracted, partially soluble digest obtained from brewer's yeast cells, preferably spent brewer's yeast cells. Digestion is accomplished by hydrolysis or autolysis of yeast cells as defined hereinbefore. Brewer's yeast autolysate contains both soluble and insoluble components derived from the whole yeast cell (e.g. Food Chemical Codex).
  • Yeast extract comprises only the water soluble components of the brewer's yeast cell, the composition of which is primarily amino-acids, peptides, carbohydrates and salts. Yeast Extract is produced through the hydrolysis of peptide bonds by the naturally occurring enzymes present in edible yeast and/or or by the addition of food-grade enzymes (Food Chemical Codex), i.e. by autolysis and/or hydrolysis as defined hereinbefore.
  • Food Chemical Codex food-grade enzymes
  • Protease is defined herein as a hydrolase acting on peptide bonds in a protein substrate in an endo-fashion, i.e. cleaving the peptide bonds anywhere in the polypeptide chain in contrast to a (exo)peptidase which is defined herein as a hydrolase acting on peptide bonds in a protein substrate in an exo fashion, i.e. acting near the ends of the polypeptide chain: aminopeptidases are cleaving off amino acids, di- tri or higher oligopeptides from the N-terminal side of the polypeptide chain and carboxypeptidases cleaving off amino acids, di- tri or higher oligopeptides form the C-terminal side of the polypeptide chain.
  • endoproteases are divided into subclasses on the basis of their catalytic mechanism: serine endoproteases (EC 3.4.21.xx), cysteine endoproteases (EC 3.4.22.xx), aspartic endoproteases (EC 3.4.23.xx) and metallo-endoproteases (EC 3.4.24.xx).
  • a "proline-specific endoprotease” is defined herein as an endoprotease cleaving protein or oligopeptides substrates at the C-terminal side of a proline residue in the protein or oligopeptides substrate.
  • the proline-specific endoprotease has been classified as EC 3.4.21.26.
  • the enzyme can be obtained from various sources such as mammalian sources, bacteria (e.f. Flavobacterium ) and fungi ( Aspergillus, in particular Aspergillus niger ) .
  • the enzyme of Aspergillus niger has been described in detail in WO02/45524 , WO02/46381 , WO03/104382 .
  • a suitable fungal enzyme from Penicillium chrysogenum is disclosed in WO2009/144269 .
  • a suitable bacterial enzyme from Flavobacterium meningosepticum is disclosed in WO03068170 .
  • Gluten is defined herein as a protein composite found in grains and is subdivided in gliadin and glutenin proteins.
  • the invention provides a process for preparing a yeast hydrolysate characterized in that the process comprises the step of contacting a gluten containing yeast hydrolysate with a proline-specific endoprotease classified as EC 3.4.21.26 of Aspergillus niger and a phosphodiesterase to produce 5'-ribonucleotides resulting in a yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm gluten, or gliadin, based on salt-free yeast dry matter and further comprising 5'-ribonucleotides 5'-GMP and / or 5'-IMP, wherein the total amount of 5'-GMP and 5'-IMP is at least 1.5% based on the NaCl free dry matter of the yeast hydrolysate.
  • a proline-specific endoprotease classified as EC 3.4.21.26 of Aspergillus niger and a phosphodiesterase
  • the resulting yeast hydrolysate is comprising less than 50 ppm gluten, more preferably less than 40 ppm, more preferably less than 30 ppm and more preferably less than 20 ppm gluten, or gliadin, based on salt-free yeast dry matter.
  • the yeast hydrolysate preferably may contain at least 5 ppm gluten, or gliadin, more preferably at least 4 ppm gluten, or gliadin, more preferably at least 3 ppm gluten, or gliadin, more preferably at least 2 ppm gluten, or gliadin.
  • the yeast hydrolysate contains at least 1 ppm gluten based on salt-free yeast dry matter.
  • the present proline-specific endoprotease provides a low gluten yeast hydrolysate wherein the yeast' RNA or derived 5'ribonucleotides is not, or not substantially, degraded and thus available for the conversion to taste enhancing 5'-ribonucleotides.
  • the process of the invention is using the proline-specific endoprotease of Aspergillus niger.
  • This enzyme is commercially available as Brewer's Clarex and may be obtained from DSM Food Specialties, Delft, The Netherlands.
  • the conditions for the step wherein the proline-specific endoprotease is used to reduce the gluten content of the yeast hydrolysate may vary from proline-specific endoprotease to proline-specific endoprotease but may be selected by the skilled person without undue burden, based on the enzymatic properties that are known in the art for the various proline-specific endoproteases.
  • the amount of proline-specific endoprotease which is contacted with the present gluten containing yeast hydrolysate is within the range of more than zero to 0.5% w/w on salt free yeast dry matter. More preferably the amount of proline-specific endoprotease which is contacted with the present gluten containing yeast hydrolysate is within the range of 0.01 to 0.5, more preferably within the range of 0.02 to 0.1 or to 0.01 w/w on salt free yeast dry matter.
  • the yeast hydrolysate may be a yeast extract or yeast autolysate as defined hereinbefore.
  • the yeast may be any suitable yeast and is preferable selected from the genera Saccharomyces, Brettanomyces, Kluyveromyces, Candida or Torula, preferably the genus Saccharomyces, more preferably Saccharomyces cerevisiae as is used as a baker's yeast or the brewer's yeast Saccharomyces carlsbergensis or its synonym Saccharomyces pastorianus as is used in the brewing industry for instance to produce lager beer.
  • the yeast is a brewer's yeast and more preferably a spent brewer's yeast which is a side product of the beer brewing process.
  • the yeast hydrolysate may be produced from yeast cells by methods well known in the art, such as by autolysis or hydrolysis as defined hereinbefore.
  • the yeast cells may contain gluten for instance because in the production process for the yeast a raw material containing gluten is used.
  • brewer's yeast more preferably spent brewer's yeast, may contain residual gluten from the barley or wheat used in the beer brewing process.
  • a yeast hydrolysate is prepared comprising less than 100 ppm gluten and at least 1 ppm, or gliadin, based on salt-free yeast dry matter.
  • the resulting yeast hydrolysate is comprising less than 50 ppm gluten, or gliadin, more preferably less than 40 ppm, more preferably less than 30 ppm and more preferably less than 20 ppm gluten, or gliadin, based on salt-free yeast dry matter.
  • the proline-specific endoprotease used in the process is reducing the amount of gluten present in the yeast hydrolysate by cleaving the gluten with the specificity as described hereinbefore for the proline-specific endoprotease.
  • the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 100 ppm otherwise, the proline-specific endoprotease is not able to reduce the gluten content.
  • the yeast hydrolysate subjected to the step with proline-specific endoprotease in order to obtain a yeast hydrolysate with less than 50 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 50 ppm and also in order to obtain a yeast hydrolysate with less than 40 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 40 ppm and also in order to obtain a yeast hydrolysate with less than 30 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 30 ppm and also in order to obtain a yeast hydrolysate with less than 20 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 20 ppm. In general, the yeast hydrolysate subjected to the step with proline-specific endoprotease
  • a proline-specific endoprotease resulting in a yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm gluten based on salt-free yeast dry matter also a phosphodiesterase to produce 5'-ribonucleotides and optionally an enzymatic treatment to convert 5'-AMP into 5'-IMP.
  • a proline-specific endoprotease to produce 5'-ribonucleotides and optionally an enzymatic treatment to convert 5'-AMP into 5'-IMP.
  • the invention provides a yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm, or gliadin, based on salt-free yeast dry matter.
  • the resulting yeast hydrolysate is comprising less than 50 ppm gluten, or gliadin, more preferably less than 40 ppm, more preferably less than 30 ppm and more preferably less than 20 ppm gluten, or gliadin, based on salt-free yeast dry matter.
  • the yeast hydrolysate preferably may contain at least 5 ppm gluten, more preferably at least 4 ppm gluten, or gliadin, more preferably at least 3 ppm gluten, or gliadin, more preferably at least 2 ppm gluten, or gliadin.
  • the yeast hydrolysate contains at least 1 ppm gluten, or gliadin, based on salt-free yeast dry matter.
  • the yeast hydrolysate may be obtainable by any suitable process, but is preferably obtainable by, or obtained by, the process of the first aspect of the invention.
  • the present yeast hydrolysate further comprises 5'-ribonucleotides.
  • the term "5'-ribonucleotides” is herein intended to refer to either the free 5'-ribonucleotides or salts thereof.
  • 5'-IMP, 5'-GMP are known for their flavour enhancing properties. They are capable of enhancing the savoury and delicious taste in certain types of food.
  • the advantage of the present yeast hydrolysate is that it provides flavour enhancing properties in combination with a low amount of gliadin and / or gluten.
  • the weight percentage of 5'-ribonucleotides in the yeast hydrolysate of the invention is based on the weight of the NaCl free dry matter of the yeast hydrolysate and is calculated as disodium salt heptahydrate (2Na.7H 2 O) of 5'-ribonucelotide.
  • the present yeast hydrolysate further comprises 5'-GMP (5'-guanine mono phosphate) and / or 5'-IMP (5'-inosine mono phosphate).
  • the present yeast hydrolysate comprises more than 60%, preferably more than 70%, more preferably more than 80%, more preferably more than 90%, more preferably more than 95% 5'-GMP and / or 5'-IMP of the amount of 5'-GMP and / or 5'-IMP which could maximally be obtained given the amount of RNA present in the yeast cell the yeast hydrolysate is obtained from.
  • the total amount of 5'-GMP and 5'-IMP in the present yeast hydrolysate is at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 5% or at least 6% based on the NaCl free dry matter of the yeast hydrolysate.
  • the yeast hydrolysate is a yeast autolysate
  • the total amount of 5'-GMP and 5'-IMP is at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 5% or at least 6% based on the NaCl free dry matter of the yeast autolysate.
  • the total amount of 5'-GMP and 5'-IMP is at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 5% or at least 6% based on the NaCl free dry matter of the yeast extract.
  • the total amount of 5'-GMP and 5'-IMP in the present yeast autolysate or yeast extract is less than 10%, preferably less than 6%, more preferably less than 5% or less than 4% based on the NaCl free dry matter of the yeast autolysate or yeast extract.
  • the yeast hydrolysate of the second aspect of the invention may be a yeast extract or yeast autolysate as defined hereinbefore.
  • the yeast may be any suitable yeast and is preferable selected from the genera Saccharomyces, Brettanomyces, Kluyveromyces, Candida or Torula, preferably the genus Saccharomyces, more preferably Saccharomyces cerevisiae as is used as a baker's yeast or the brewer's yeast Saccharomyces carlsbergensis or its synonym Saccharomyces pastorianus as is used in the brewing industry for instance to produce lager beer.
  • the yeast is a brewer's yeast and more preferably a spent brewer's yeast which is a side product of the beer brewing process.
  • the invention provides the use of the yeast hydrolysate or the use of the yeast extract or a yeast autolysate of the second aspect of the invention in a process flavour reaction, in meat applications, or as a flavour enhancer, or as a flavour improver, or in a top note carrier or in a table-top application.
  • the invention provides a flavour enhancer, a meat product, a flavour improver, a top-note carrier or a table-top application which comprises the yeast hydrolysate or the yeast extract or the yeast autolysate of the second aspect of the invention.
  • the amount of gluten in the yeast hydrolysate was determined by UPEX-extraction (Universal Prolamin and Glutelin Extractant solution) of the gluten/gliadin and quantifying by an ELISA-test essentially as described by M.C. Mena et al. Talanta 91 (2012) pp 33-40 "Comprehensive analysis of gluten in processed foods using a new extraction method and a competitive ELISA based on the R5 antibody ".
  • the gluten extraction procedure is based on reducing Tris (2-carboxyethyl)-phosphine (TCEP) (Sigma-Aldrich art nr.: C4706,The Netherlands) and anionic surfactant N-lauroylsarcosine (Sarkosyl) (Sigma-Aldrich art nr.: 61745, The Netherlands) reagents in PBS (Sigma-Aldrich art nr.: P3813, The Netherlands).
  • TCEP Tris (2-carboxyethyl)-phosphine
  • Sarkosyl anionic surfactant N-lauroylsarcosine
  • Spent brewer's cream yeast was sourced from a brewery in the Netherlands. The obtained yeast was heated to 55°C and the pH was set to 5.3.
  • Alcalase® (Alcalase® contains the endoprotease subtilisin Carlsberg of Bacillus licheniformis and is produced by Novozymes, Denmark and purchased from Sigma, catalogue number P4860) was added to the slurry at a 1% w/w dosage on yeast salt free dry matter. The suspension was incubated overnight. After Alcalase® incubation the suspension was set to pH 5.3 and divided into 4 different fractions. A proline-specific endoprotease was dosed in a range of 0 to 0.5 % w/w on salt free dry matter, according to table 1.
  • the suspensions were maintained at 61°C and incubated for 15 hours. After incubation the suspensions were heated to stop residual enzyme activity. Samples of the suspensions and extracts (after clarification of suspensions) were taken and concentrated by evaporation. The concentrates were analysed for gliadin content by means the UPEX extraction and ELISA test as described in the Materials and Methods.
  • a brewer's yeast extract (EXPRESA 2200S, DSM FOOD SPECIALTIES, Delft, The Netherlands) in powder formulation was dissolved in water to 10% w/w.
  • the pH of the solution was set to 5.3 and the temperature was maintained at 61 degrees centigrade.
  • the sample was divided into two fractions.
  • a proline-specific endoprotease was added to one of the 2 flasks containing the yeast extract and was dosed at 0.5% w/w on salt free yeast dry matter. The solution was incubated for 15 hours. After incubation the solution was heated to 90°C to stop residual enzyme activity. The obtained solution was concentrated by evaporation of water. A concentrated sample was used were analysed for gliadin content by means the UPEX extraction and ELISA test as described in the Materials and Methods. Table 2: Effect of proline-specific endoprotease Sample Gliadin content ppm on total dry matter (w/w) Control (without enzyme) 200 0.5% w/w proline-specific endoprotease ⁇ 20
  • RNA after reducing the gliadin content with proline-specific endoprotease is compared with treatment with commercially available proteases which are also suitable for reduction of gliadin content.
  • a brewer's yeast acquired from a Dutch brewery was used to produce a nucleotide containing yeast autolysate with a high yeast solubilisation yield.
  • Yeast RNA was measured and was 3.4% on dry matter. Firstly, the yeast was heat shocked 5 min at 95°C. Dry matter was subsequently dissolved by two protease steps.
  • Alcalase® contains the endoprotease subtilisin Carlsberg of Bacillus licheniformis and is produced by Novozymes, Denmark and purchased from Sigma, catalogue number P4860
  • pH 8 pH 8
  • P4860 pH 8
  • 62 °C temperature of 62 °C during 6 hours
  • proteases were separately applied in different experiments (1% on dry matter) and incubated at a pH of 5.2, with a temperature of 51.5°C during 15 hours. After these two protease steps the enzymes were inactivated by 85°C heat shock. RNA was converted by incubation with 5'-phosphodiesterase (DSM) and deaminase (Amano) to produce 5'GMP and 5'IMP. The incubation was at pH 5.3 and temperature at 60°C during 15 hours. After these different processes the amount of 5'-GMP and 5'-IMP in the samples (expressed as weight percentage of the disodium heptahydrate thereof based on sodium chloride free dry matter) were subsequently determined by means of HPLC according to the following method.
  • DSM 5'-phosphodiesterase
  • Mano deaminase
  • 5'-GMP and 5'-IMP were quantified by HPLC using a Whatman Partisil 10-SAX column, a phosphate buffer pH 3.35 as eluent and UV detection. Concentrations were calculated on basis of 5'-GMP and 5'-IMP standards.
  • Sodium chloride was determined by measuring the chloride ions in the sample with a Jenway chloride meter PCLM 3 (Jenway, Essex, England) and calculating the corresponding amount of sodium chloride.
  • Table 3 shows that using a proline-specific endoprotease provides at least 90% 5'-GMP and 5'-IMP of the expected amount 5'-GMP and 5'-IMP, given the RNA content of the starting yeast (3.4%).
  • the proline-specific endoprotease is able to reduce gluten as shown in example 1 and 2, while the RNA present in the yeast is not degraded and thus available to be converted into 5'-ribonucleotides like 5'-GMP and 5'-IMP. This is surprising in view of the amount of 5'-GMP and 5'-IMP for the other proteases which apparently also degraded the RNA of the yeast.

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Description

    Field of the invention
  • The present invention relates to low gluten yeast hydrolysates and a process for the for producing the same using a prolyl-specific endoprotease.
    • Background of the invention
  • For the production of yeast extract, typically baker's and torula yeast cells may be subjected to autolysis. Some sources of yeast (e.g. spent brewer's yeast) contain residues of gluten (gliadins). As a consequence, extracts and suspensions derived of yeasts sources containing gliadins have limited use in food applications due to the intolerance towards gliadins in part of the population (Celiac disease). It was observed that typical yeast extracts or autolysates obtained from sources containing gliadins (e.g. brewer's yeast extracts) contain gliadins in a concentration exceeding the acceptable threshold of 20 ppm by weight.
  • Reduction of gliadins from yeast sources containing gliadins enables the use of these yeasts and extracts thereof is food applications which require absence of low concentrations of gluten.
  • A problem with the reduction of gliadins in yeast is to reduce specifically the gliadin content without undesired side effects of the treatment. For example, it is undesired that the treatment aiming to reduce the gliadin content also degrades the yeast' RNA.
  • US5286630 discloses yeast extracts from Candida utilis which can be used as flavor enhancers and emulsifiers. The extract is isolated by heating Candida utilis cells, rupturing the cells, separating the ruptured and recovering the soluble yeast extract.
  • WO2005/027953 discloses the use of proline specific endoproteases to hydrolyse peptides and proteins comprising 4 to 40 amino acid residues.
  • WO2005/067734 discloses a process to produce a composition containing 5'-ribonucleotides wherein a microorganism is subjected to autolysis under conditions at which a substantial part of the RNA remains in a form degradable into 5'-ribonucleotides and at which a substantial part of the RNA remains associated with the cell wall fraction.
  • WO2012/110534 discloses a process to produce a yeast extract comprising a) subjecting yeast to autolysis, and b) subjecting the autolysate to solid/liquid separation and recovering the soluble fraction, whereby the solid liquid separation in step b) is done at a pH of less than 5.1.
  • JP2007 049988 discloses the provision of yeast extracts by the use of a protease, a 5'-phosphodiesterase and 5'-adelyatedeaminase, resulting in an increase in the peptide and nucleic acid content of the hydrolysates.
  • EP2256184 describes a Saccharomyces cerevisiae mutant strain having a RNA content of 14% by weight or more based on the dry cell weight and a method for producing a yeast of high RNA content by using the strain.
  • It was now surprisingly found that gliadins can be removed from a yeast source containing gliadins by incubation with a proline-specific endoprotease during the hydrolysis and/or autolysis process. It was surprisingly found that the gliadins can effectively be hydrolyzed to a level below 20 ppm by weight on dry matter content of the yeast autolysates and extracts. Further, it was found that incubation with a proline-specific endoprotease during the hydrolysis and/or autolysis process did not degrade the RNA present in the yeast.
    • Definitions
  • "Yeast" is defined herein as a solid, paste or liquid composition comprising yeast cells. Preferably the yeast cells are from the genus Saccharomyces. The yeast may be produced in a fermentation process such as a process for the production common baker's yeast. Preferably the yeast is a brewer's yeast, such as a spent brewer's yeast that may be obtained as a side stream from beer brewing processes.
  • "Autolysis" is defined herein as the enzymatic breakdown of yeast cells using endogenous yeast enzymes and optionally exogenous added enzymes. Autolysis may result in both a yeast autolysate and a yeast extract (see definitions below).
  • "Hydrolysis" is defined herein as the enzymatic breakdown of yeast cells using only exogenous enzymes. The endogenous yeast enzymes are first inactivated for instance by a heat shock. Hydrolysis may also result in both a yeast autolysate and a yeast extract (see definitions below).
  • "Yeast hydrolysate" is defined herein as the digest of yeast, such as a spent brewer's yeast, obtained by autolysis or hydrolysis as defined herein before and resulting in a yeast autolysate or a yeast extract as defined herein below.
  • "Yeast autolysate" is the concentrated, not extracted, partially soluble digest obtained from brewer's yeast cells, preferably spent brewer's yeast cells. Digestion is accomplished by hydrolysis or autolysis of yeast cells as defined hereinbefore. Brewer's yeast autolysate contains both soluble and insoluble components derived from the whole yeast cell (e.g. Food Chemical Codex).
  • "Yeast extract" comprises only the water soluble components of the brewer's yeast cell, the composition of which is primarily amino-acids, peptides, carbohydrates and salts. Yeast Extract is produced through the hydrolysis of peptide bonds by the naturally occurring enzymes present in edible yeast and/or or by the addition of food-grade enzymes (Food Chemical Codex), i.e. by autolysis and/or hydrolysis as defined hereinbefore.
  • "Protease" is defined herein as a hydrolase acting on peptide bonds in a protein substrate in an endo-fashion, i.e. cleaving the peptide bonds anywhere in the polypeptide chain in contrast to a (exo)peptidase which is defined herein as a hydrolase acting on peptide bonds in a protein substrate in an exo fashion, i.e. acting near the ends of the polypeptide chain: aminopeptidases are cleaving off amino acids, di- tri or higher oligopeptides from the N-terminal side of the polypeptide chain and carboxypeptidases cleaving off amino acids, di- tri or higher oligopeptides form the C-terminal side of the polypeptide chain. The endoproteases are divided into subclasses on the basis of their catalytic mechanism: serine endoproteases (EC 3.4.21.xx), cysteine endoproteases (EC 3.4.22.xx), aspartic endoproteases (EC 3.4.23.xx) and metallo-endoproteases (EC 3.4.24.xx).
  • A "proline-specific endoprotease" is defined herein as an endoprotease cleaving protein or oligopeptides substrates at the C-terminal side of a proline residue in the protein or oligopeptides substrate. The proline-specific endoprotease has been classified as EC 3.4.21.26. The enzyme can be obtained from various sources such as mammalian sources, bacteria (e.f. Flavobacterium) and fungi (Aspergillus, in particular Aspergillus niger). The enzyme of Aspergillus niger has been described in detail in WO02/45524 , WO02/46381 , WO03/104382 . A suitable fungal enzyme from Penicillium chrysogenum is disclosed in WO2009/144269 . A suitable bacterial enzyme from Flavobacterium meningosepticum is disclosed in WO03068170 .
  • "Gluten" is defined herein as a protein composite found in grains and is subdivided in gliadin and glutenin proteins.
    • Detailed description of the invention
  • In a first aspect, the invention provides a process for preparing a yeast hydrolysate characterized in that the process comprises the step of contacting a gluten containing yeast hydrolysate with a proline-specific endoprotease classified as EC 3.4.21.26 of Aspergillus niger and a phosphodiesterase to produce 5'-ribonucleotides resulting in a yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm gluten, or gliadin, based on salt-free yeast dry matter and further comprising 5'-ribonucleotides 5'-GMP and / or 5'-IMP, wherein the total amount of 5'-GMP and 5'-IMP is at least 1.5% based on the NaCl free dry matter of the yeast hydrolysate. Preferably, the resulting yeast hydrolysate is comprising less than 50 ppm gluten, more preferably less than 40 ppm, more preferably less than 30 ppm and more preferably less than 20 ppm gluten, or gliadin, based on salt-free yeast dry matter. The yeast hydrolysate preferably may contain at least 5 ppm gluten, or gliadin, more preferably at least 4 ppm gluten, or gliadin, more preferably at least 3 ppm gluten, or gliadin, more preferably at least 2 ppm gluten, or gliadin. The yeast hydrolysate contains at least 1 ppm gluten based on salt-free yeast dry matter. The inventors of the present invention surprisingly found that the present proline-specific endoprotease provides a low gluten yeast hydrolysate wherein the yeast' RNA or derived 5'ribonucleotides is not, or not substantially, degraded and thus available for the conversion to taste enhancing 5'-ribonucleotides.
  • The process of the invention is using the proline-specific endoprotease of Aspergillus niger. This enzyme is commercially available as Brewer's Clarex and may be obtained from DSM Food Specialties, Delft, The Netherlands.
  • The conditions for the step wherein the proline-specific endoprotease is used to reduce the gluten content of the yeast hydrolysate, such as pH and temperature, may vary from proline-specific endoprotease to proline-specific endoprotease but may be selected by the skilled person without undue burden, based on the enzymatic properties that are known in the art for the various proline-specific endoproteases.
  • Preferably, the amount of proline-specific endoprotease which is contacted with the present gluten containing yeast hydrolysate is within the range of more than zero to 0.5% w/w on salt free yeast dry matter. More preferably the amount of proline-specific endoprotease which is contacted with the present gluten containing yeast hydrolysate is within the range of 0.01 to 0.5, more preferably within the range of 0.02 to 0.1 or to 0.01 w/w on salt free yeast dry matter.
  • The yeast hydrolysate may be a yeast extract or yeast autolysate as defined hereinbefore. The yeast may be any suitable yeast and is preferable selected from the genera Saccharomyces, Brettanomyces, Kluyveromyces, Candida or Torula, preferably the genus Saccharomyces, more preferably Saccharomyces cerevisiae as is used as a baker's yeast or the brewer's yeast Saccharomyces carlsbergensis or its synonym Saccharomyces pastorianus as is used in the brewing industry for instance to produce lager beer. In a preferred embodiment the yeast is a brewer's yeast and more preferably a spent brewer's yeast which is a side product of the beer brewing process.
  • The yeast hydrolysate may be produced from yeast cells by methods well known in the art, such as by autolysis or hydrolysis as defined hereinbefore. The yeast cells may contain gluten for instance because in the production process for the yeast a raw material containing gluten is used. In particular brewer's yeast, more preferably spent brewer's yeast, may contain residual gluten from the barley or wheat used in the beer brewing process.
  • In the process of the invention a yeast hydrolysate is prepared comprising less than 100 ppm gluten and at least 1 ppm, or gliadin, based on salt-free yeast dry matter. Preferably, the resulting yeast hydrolysate is comprising less than 50 ppm gluten, or gliadin, more preferably less than 40 ppm, more preferably less than 30 ppm and more preferably less than 20 ppm gluten, or gliadin, based on salt-free yeast dry matter. The proline-specific endoprotease used in the process is reducing the amount of gluten present in the yeast hydrolysate by cleaving the gluten with the specificity as described hereinbefore for the proline-specific endoprotease. The skilled person will easily understand that in order to obtain a yeast hydrolysate with less than 100 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 100 ppm otherwise, the proline-specific endoprotease is not able to reduce the gluten content. Likewise, in order to obtain a yeast hydrolysate with less than 50 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 50 ppm and also in order to obtain a yeast hydrolysate with less than 40 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 40 ppm and also in order to obtain a yeast hydrolysate with less than 30 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 30 ppm and also in order to obtain a yeast hydrolysate with less than 20 ppm gluten, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more than 20 ppm. In general, the yeast hydrolysate subjected to the step with proline-specific endoprotease must contain more gluten than the yeast hydrolysate obtained after the step wherein the proline-specific endoprotease is reducing the gluten content.
  • It is included in the present step of contacting a gluten containing yeast hydrolysate with a proline-specific endoprotease resulting in a yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm gluten based on salt-free yeast dry matter also a phosphodiesterase to produce 5'-ribonucleotides and optionally an enzymatic treatment to convert 5'-AMP into 5'-IMP. The advantage of using a combination of a phosphodiesterase with the present proline-specific endoprotease is that a low gluten yeast hydrolysate is obtained with increased amounts of 5'-ribonucleotides.
  • In a second aspect the invention provides a yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm, or gliadin, based on salt-free yeast dry matter. Preferably, the resulting yeast hydrolysate is comprising less than 50 ppm gluten, or gliadin, more preferably less than 40 ppm, more preferably less than 30 ppm and more preferably less than 20 ppm gluten, or gliadin, based on salt-free yeast dry matter. The yeast hydrolysate preferably may contain at least 5 ppm gluten, more preferably at least 4 ppm gluten, or gliadin, more preferably at least 3 ppm gluten, or gliadin, more preferably at least 2 ppm gluten, or gliadin. The yeast hydrolysate contains at least 1 ppm gluten, or gliadin, based on salt-free yeast dry matter. The yeast hydrolysate may be obtainable by any suitable process, but is preferably obtainable by, or obtained by, the process of the first aspect of the invention.
  • The present yeast hydrolysate further comprises 5'-ribonucleotides. The term "5'-ribonucleotides" is herein intended to refer to either the free 5'-ribonucleotides or salts thereof. 5'-IMP, 5'-GMP are known for their flavour enhancing properties. They are capable of enhancing the savoury and delicious taste in certain types of food. Thus the advantage of the present yeast hydrolysate is that it provides flavour enhancing properties in combination with a low amount of gliadin and / or gluten.
  • The weight percentage of 5'-ribonucleotides in the yeast hydrolysate of the invention (%w/w) is based on the weight of the NaCl free dry matter of the yeast hydrolysate and is calculated as disodium salt heptahydrate (2Na.7H2O) of 5'-ribonucelotide.
  • In a further preferred embodiment of the first or second aspect, the present yeast hydrolysate further comprises 5'-GMP (5'-guanine mono phosphate) and / or 5'-IMP (5'-inosine mono phosphate). Preferably, the present yeast hydrolysate comprises more than 60%, preferably more than 70%, more preferably more than 80%, more preferably more than 90%, more preferably more than 95% 5'-GMP and / or 5'-IMP of the amount of 5'-GMP and / or 5'-IMP which could maximally be obtained given the amount of RNA present in the yeast cell the yeast hydrolysate is obtained from.
  • Preferably, the total amount of 5'-GMP and 5'-IMP in the present yeast hydrolysate is at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 5% or at least 6% based on the NaCl free dry matter of the yeast hydrolysate. If the yeast hydrolysate is a yeast autolysate, the total amount of 5'-GMP and 5'-IMP is at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 5% or at least 6% based on the NaCl free dry matter of the yeast autolysate. If the present yeast hydrolysate is a yeast extract, the total amount of 5'-GMP and 5'-IMP is at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5, 4%, 5% or at least 6% based on the NaCl free dry matter of the yeast extract. Preferably, the total amount of 5'-GMP and 5'-IMP in the present yeast autolysate or yeast extract is less than 10%, preferably less than 6%, more preferably less than 5% or less than 4% based on the NaCl free dry matter of the yeast autolysate or yeast extract.
  • The yeast hydrolysate of the second aspect of the invention may be a yeast extract or yeast autolysate as defined hereinbefore. The yeast may be any suitable yeast and is preferable selected from the genera Saccharomyces, Brettanomyces, Kluyveromyces, Candida or Torula, preferably the genus Saccharomyces, more preferably Saccharomyces cerevisiae as is used as a baker's yeast or the brewer's yeast Saccharomyces carlsbergensis or its synonym Saccharomyces pastorianus as is used in the brewing industry for instance to produce lager beer. In a preferred embodiment the yeast is a brewer's yeast and more preferably a spent brewer's yeast which is a side product of the beer brewing process.
  • In a third aspect the invention provides the use of the yeast hydrolysate or the use of the yeast extract or a yeast autolysate of the second aspect of the invention in a process flavour reaction, in meat applications, or as a flavour enhancer, or as a flavour improver, or in a top note carrier or in a table-top application.
  • In a fourth aspect the invention provides a flavour enhancer, a meat product, a flavour improver, a top-note carrier or a table-top application which comprises the yeast hydrolysate or the yeast extract or the yeast autolysate of the second aspect of the invention.
    • MATERIALS AND METHODS Gluten determination
  • The amount of gluten in the yeast hydrolysate was determined by UPEX-extraction (Universal Prolamin and Glutelin Extractant solution) of the gluten/gliadin and quantifying by an ELISA-test essentially as described by M.C. Mena et al. Talanta 91 (2012) pp 33-40 "Comprehensive analysis of gluten in processed foods using a new extraction method and a competitive ELISA based on the R5 antibody ".
  • The gluten extraction procedure is based on reducing Tris (2-carboxyethyl)-phosphine (TCEP) (Sigma-Aldrich art nr.: C4706,The Netherlands) and anionic surfactant N-lauroylsarcosine (Sarkosyl) (Sigma-Aldrich art nr.: 61745, The Netherlands) reagents in PBS (Sigma-Aldrich art nr.: P3813, The Netherlands).
    1. 1. 250 mg dry yeast hydrolysate was weighed and transferred to a 10-ml polypropylene tube.
    2. 2. 2.5 ml UPEX solution (5 mM TCEP, 2% N-lauroylsarcosine in PBS, pH 7) was added to the tube containing the yeast hydrolysate. In order to prevent inactivation of the reducing agent, UPEX solution was prepared immediately before use.
    3. 3. The tubes were closed tightly and the caps were covered with film to avoid evaporation.
    4. 4. The contents of the tubes were mixed thoroughly by vortexing for 5 to 10 seonds and the tubes were placed in a rack.
    5. 5. The tubes were incubated in a GL water bath type: 1003 at 50°C for 40 minutes.
    6. 6. The tubes were allowed to cool for 5 min at room temperature.
    7. 7. 7.5 ml of 80% ethanol/water (v/v) was added and the samples were thoroughly dispersed by vortexing for 10-60 seconds until total dispersion of the sample was achieved and then incubated for 1 h at room temperature in a rotary (head-overhead) shaker (Stuart Scientific Rollermixer type: SRT2) at 45 turns/min.
    8. 8. The tubes were centrifuged in a bench-top centrifuge (Eppendorf model 5810R) for 10 min at 2500 g at room temperature.
    9. 9. Using fresh Pasteur pipettes, the supernatant from each tube was transferred to a clean 10-ml polypropylene tube.
    10. 10. The solution was analysed by the ELISA test within 24 h of extraction.
  • For the ELISA test the Ridascreen® Gliadin Competitive kit supplied by R-Biopharm) was used (R-Biopharm AG, Darmstadt, Germany).
    • EXAMPLES Example 1 Reduction of gliadin
  • Spent brewer's cream yeast was sourced from a brewery in the Netherlands. The obtained yeast was heated to 55°C and the pH was set to 5.3. Alcalase® (Alcalase® contains the endoprotease subtilisin Carlsberg of Bacillus licheniformis and is produced by Novozymes, Denmark and purchased from Sigma, catalogue number P4860) was added to the slurry at a 1% w/w dosage on yeast salt free dry matter. The suspension was incubated overnight. After Alcalase® incubation the suspension was set to pH 5.3 and divided into 4 different fractions. A proline-specific endoprotease was dosed in a range of 0 to 0.5 % w/w on salt free dry matter, according to table 1. The suspensions were maintained at 61°C and incubated for 15 hours. After incubation the suspensions were heated to stop residual enzyme activity. Samples of the suspensions and extracts (after clarification of suspensions) were taken and concentrated by evaporation. The concentrates were analysed for gliadin content by means the UPEX extraction and ELISA test as described in the Materials and Methods. Table 1: Dose-effect of proline-specific endoprotease
    Sample Gliadin content ppm on total dry matter (w/w)
    Suspension, no enzyme dosage 170
    Suspension, 0.05% w/w enzyme dosage 20
    Suspension, 0.25% w/w enzyme dosage <20
    Suspension, 0.50% w/w enzyme dosage <20
    Extract, no enzyme dosage 220
    Extract, 0.05% w/w enzyme added 25
    Extract, 0.25% w/w enzyme added <20
    Extract, 0.50% w/w enzyme added <20
    • Example 2 Reduction of gliadin
  • A brewer's yeast extract (EXPRESA 2200S, DSM FOOD SPECIALTIES, Delft, The Netherlands) in powder formulation was dissolved in water to 10% w/w. The pH of the solution was set to 5.3 and the temperature was maintained at 61 degrees centigrade. The sample was divided into two fractions.
  • A proline-specific endoprotease was added to one of the 2 flasks containing the yeast extract and was dosed at 0.5% w/w on salt free yeast dry matter. The solution was incubated for 15 hours. After incubation the solution was heated to 90°C to stop residual enzyme activity. The obtained solution was concentrated by evaporation of water. A concentrated sample was used were analysed for gliadin content by means the UPEX extraction and ELISA test as described in the Materials and Methods. Table 2: Effect of proline-specific endoprotease
    Sample Gliadin content ppm on total dry matter (w/w)
    Control (without enzyme) 200
    0.5% w/w proline-specific endoprotease <20
    • Example 3 Reduction of RNA of proline-specific endoprotease compared with proteases
  • In this example the undesired degradation of RNA after reducing the gliadin content with proline-specific endoprotease is compared with treatment with commercially available proteases which are also suitable for reduction of gliadin content. A brewer's yeast acquired from a Dutch brewery was used to produce a nucleotide containing yeast autolysate with a high yeast solubilisation yield. Yeast RNA was measured and was 3.4% on dry matter. Firstly, the yeast was heat shocked 5 min at 95°C. Dry matter was subsequently dissolved by two protease steps. First Alcalase® (Alcalase® contains the endoprotease subtilisin Carlsberg of Bacillus licheniformis and is produced by Novozymes, Denmark and purchased from Sigma, catalogue number P4860) was incubated at pH 8 and a temperature of 62 °C during 6 hours (dosage:0.8% on dry matter). For the second protease treatment several different enzymes were tested:
    • Proteax (available from Amano Enzyme)
    • Peptidase R (available from Amano Enzyme)
    • Accelerzyme (available from DSM)
    • Sumizyme FP (available from Shin Nihon)
    • proline-specific endoprotease (Brewers Clarex, from DSM)
  • These proteases were separately applied in different experiments (1% on dry matter) and incubated at a pH of 5.2, with a temperature of 51.5°C during 15 hours. After these two protease steps the enzymes were inactivated by 85°C heat shock. RNA was converted by incubation with 5'-phosphodiesterase (DSM) and deaminase (Amano) to produce 5'GMP and 5'IMP. The incubation was at pH 5.3 and temperature at 60°C during 15 hours. After these different processes the amount of 5'-GMP and 5'-IMP in the samples (expressed as weight percentage of the disodium heptahydrate thereof based on sodium chloride free dry matter) were subsequently determined by means of HPLC according to the following method. 5'-GMP and 5'-IMP were quantified by HPLC using a Whatman Partisil 10-SAX column, a phosphate buffer pH 3.35 as eluent and UV detection. Concentrations were calculated on basis of 5'-GMP and 5'-IMP standards. Sodium chloride was determined by measuring the chloride ions in the sample with a Jenway chloride meter PCLM 3 (Jenway, Essex, England) and calculating the corresponding amount of sodium chloride.
  • The effectiveness of the proteases was measured by determination of solubilisation yield and degree of hydrolysis of the protein. The results are summarized in table 3: Table 3:
    Applied enzyme for second protease step 5'GMP (2Na7H2O in autoloysate on dm) 5'IMP (2Na7H2O in autoloysate on dm) Solubilisation yield1 Degree of hydrolysis protein2
    (%) (%) (%) (%)
    Accelerzyme 0,48 0.4 55 26
    Proteax 0,08 0.25 59 47
    Validase FP 0 0 64 34
    Peptidase R 0,59 0 64 29
    Brewers clarex 0.9 1.0 60 14
    Expected 1.0 1.1
    1. Calculated by DM supernatant/DM broth
    2. Calculated by Free amino acids/total amino acids after acid hydrolysis measured by HPLC
  • Table 3 shows that using a proline-specific endoprotease provides at least 90% 5'-GMP and 5'-IMP of the expected amount 5'-GMP and 5'-IMP, given the RNA content of the starting yeast (3.4%). Thus the proline-specific endoprotease is able to reduce gluten as shown in example 1 and 2, while the RNA present in the yeast is not degraded and thus available to be converted into 5'-ribonucleotides like 5'-GMP and 5'-IMP. This is surprising in view of the amount of 5'-GMP and 5'-IMP for the other proteases which apparently also degraded the RNA of the yeast.

Claims (14)

  1. A process for preparing a yeast hydrolysate characterized in that the process comprises the step of contacting a gluten containing yeast hydrolysate with a proline-specific endoprotease classified as EC 3.4.21.26 of Aspergillus niger and a phosphodiesterase to produce 5'-ribonucleotides resulting in a yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm gluten based on salt-free yeast dry matter and further comprising 5'-ribonucleotides 5'-GMP and / or 5'-IMP, wherein the total amount of 5'-GMP and 5'-IMP is at least 1.5% based on the NaCl free dry matter of the yeast hydrolysate.
  2. A process according to claim 1, wherein the resulting yeast hydrolysate is comprising less than 50 ppm gluten, more preferably less than 40 ppm, more preferably less than 30 ppm and more preferably less than 20 ppm gluten based on salt-free yeast dry matter.
  3. A process according to any of the preceding claims wherein the yeast hydrolysate is a yeast extract or yeast autolysate.
  4. A process according to any of the preceding claims wherein the yeast hydrolysate is a spent brewer's yeast hydrolysate.
  5. Process according to any of the preceding claims, wherein the yeast is a species from the genera Saccharomyces, Brettanomyces, Kluyveromyces, Candida or Torula, preferably the genus Saccharomyces.
  6. A yeast hydrolysate comprising less than 100 ppm gluten and at least 1 ppm gluten based on salt-free yeast dry matter, further comprising 5'-ribonucleotides.
  7. A yeast hydrolysate according to claim 6, comprising less than 50 ppm gluten and at least 1 ppm gluten based on salt-free yeast dry matter, further comprising 5'-ribonucleotides.
  8. A yeast hydrolysate according to claim 6, comprising less 40 ppm and at least 1 ppm gluten based on salt-free yeast dry matter, further comprising 5'-ribonucleotides.
  9. A yeast hydrolysate according to claim 6 comprising less 30 ppm and at least 1 ppm gluten based on salt-free yeast dry matter, further comprising 5'-ribonucleotides.
  10. A yeast hydrolysate according to claim 6, comprising less than 20 ppm gluten and at least 1 ppm gluten based on salt-free yeast dry matter, further comprising 5'-ribonucleotides.
  11. A yeast hydrolysate according to any of the claims 6 to 10, further comprising 5'-GMP and 5'-IMP, preferably the total amount 5'-GMP and 5'-IMP is at least 2% based on the NaCl free dry matter of the yeast hydrolysate.
  12. A yeast hydrolysate according to any of the claims 6 to 11, wherein the yeast hydrolysate is a yeast extract or a yeast autolysate.
  13. Use of the yeast hydrolysate of any of the claims 6 to 11 or the yeast extract or a yeast autolysate of claim 12 in a process flavour reaction, in meat applications, or in a flavour enhancer, or in a flavour improver, or in a top note carrier or in a table-top application.
  14. A flavour enhancer, a meat product, a flavour improver, a top-note carrier or a table-top application which comprises the yeast hydrolysate of any of the claims 6 to 11, or the yeast extract or a yeast autolysate of claim 12.
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Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220046966A1 (en) * 2018-12-13 2022-02-17 Dsm Ip Assets B.V. Umami flavor composition
RU2724972C1 (en) * 2019-11-27 2020-06-29 Федеральное государственное бюджетное образовательное учреждение высшего образования "Томский государственный университет систем управления и радиоэлектроники" (ТУСУР) Meander microstrip delay line of two turns, which protects against ultrashort pulses
RU2732805C1 (en) * 2019-11-27 2020-09-22 Федеральное государственное бюджетное образовательное учреждение высшего образования "Томский государственный университет систем управления и радиоэлектроники" (ТУСУР) Modified microstrip line protecting from ultrashort pulses
RU2724970C1 (en) * 2019-11-27 2020-06-29 Федеральное государственное бюджетное образовательное учреждение высшего образования "Томский государственный университет систем управления и радиоэлектроники" (ТУСУР) Meander line delay with face communication of two turns, which protects from ultrashort pulses
RU2724983C1 (en) * 2019-12-09 2020-06-29 Федеральное государственное бюджетное образовательное учреждение высшего образования "Томский государственный университет систем управления и радиоэлектроники" (ТУСУР) Improved meander delay line with face connection, which protects from ultrashort pulses
CN113862322B (en) * 2021-11-15 2024-05-14 唐山拓普生物科技有限公司 Preparation method and application of beer yeast polypeptide with high antioxidant activity

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007049988A (en) * 2005-07-20 2007-03-01 Nippon Paper Chemicals Co Ltd Yeast extract and method for producing the same
WO2009007424A1 (en) * 2007-07-10 2009-01-15 Dsm Ip Assets B.V. Yeast autolysates
US20090324778A1 (en) * 2006-09-12 2009-12-31 Lesaffre Et Compagnie Novel preparation of phosphodiesterase of plant origin

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5286630A (en) * 1991-12-05 1994-02-15 Phillips Petroleum Company Yeast extracts from candida utilis and the production of same
KR20030085112A (en) * 2000-12-07 2003-11-03 디에스엠 아이피 어셋츠 비.브이. Protein hydrolysates enriched in peptides having a carboxy terminal proline residue
WO2003104382A1 (en) * 2002-06-07 2003-12-18 Dsm Ip Assets B.V. Improved method for the prevention or reduction of haze in beverages
ES2746173T3 (en) 2003-09-23 2020-03-05 Dsm Ip Assets Bv Use of proline-specific endoproteases to hydrolyze peptides and proteins
US8084068B2 (en) 2004-01-09 2011-12-27 Dsm Ip Assets B.V. Process for the production of compositions containing ribonucleotides and their use as flavouring agents
EP1657300A1 (en) * 2004-11-10 2006-05-17 N-Zyme BioTec GmbH Beverages having reduced prolamine content and their preparation method
RU2446210C2 (en) * 2006-02-02 2012-03-27 ДСМ АйПи АССЕТС Б.В. Food product containing proline-specific protease, its production method and its application for splitting toxic and allergenic gluten peptides
JP5102076B2 (en) * 2008-03-06 2012-12-19 アサヒグループホールディングス株式会社 A Saccharomyces cerevisiae mutant and a method for producing a yeast with a high RNA content using the mutant.
AU2012217128B2 (en) * 2011-02-17 2015-10-01 Dsm Ip Assets B.V. Process for the production of yeast extracts having low turbidity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007049988A (en) * 2005-07-20 2007-03-01 Nippon Paper Chemicals Co Ltd Yeast extract and method for producing the same
US20090324778A1 (en) * 2006-09-12 2009-12-31 Lesaffre Et Compagnie Novel preparation of phosphodiesterase of plant origin
WO2009007424A1 (en) * 2007-07-10 2009-01-15 Dsm Ip Assets B.V. Yeast autolysates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 200721, Derwent World Patents Index; AN 2007-203803 *
EDENS L ET AL: "Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, AMERICAN CHEMICAL SOCIETY, BOOKS AND JOURNALS DIVISION, vol. 53, no. 20, 9 September 2005 (2005-09-09), pages 7950 - 7957, XP002374542, ISSN: 0021-8561, DOI: 10.1021/JF050652C *

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